1. Field of the Invention
This invention relates generally to disease diagnosis and to the identification of disease carriers. More specifically, the present invention provides methods for identifying individuals who are afflicted with or carriers of diseases associated with one or more genomic deletion.
2. Description of the Related Art
Van Buchem""s disease (VBD) is a rare autosomal recessive disorder that results in a bone dysplasia referred to as craniotubular hyperostosis. VBD was first described in 1962 as including osteosclerosis of the skull, mandible, clavicles, ribs, and diaphysis of the long bones beginning during puberty and, in some cases, leading to optic atrophy and perceptive deafness from nerve pressure. Van Buchem et al., Am. J. Med. 33:387-397 (1962).
More recently, additional occurrences of VBD have been reported. In 1988, Fryns et al. described a 7.5-year-old boy with VBD. This patient had presented at 2 months of age with left-side peripheral facial nerve palsy but had, at that time, no radiologically visible signs of sclerosis of the skull. Europ. J. Pediat. 147:99-100 (1988).
In 1997, Balemans et al. studied 11 VBD patients from a highly inbred and geophraphically isolated Dutch family. Each of these patients shared a common ancestor from 9 preceding generations. By applying a genome wide search for linkage using more than 300 microsatellite markers having an average spacing of 10 cM, these authors found a maximum lod score of 9.33 at theta=0.01 with marker D17S1299 and narrowed the assignment to a region of less than 1 cM between markers D17S1787 and D17S934. Am. J. Hum. Genet. 61(Suppl.):A12 (1997); See, also, Van Hul et al., Am. J. Hum. Genet. 62:391-399 (1998).
A related disease sclerosteosis is an autosomal semi-dominant disease that shares some of the clinical symptoms of VBD. The term xe2x80x9csclerosteosisxe2x80x9d has been applied to a disorder similar to Van Buchem hyperostosis corticalis generalisata but differing in the radiologic appearance of the bone changes and in the presence of asymmetric cutaneous syndactyly of the index and middle fingers in many cases. In Handbuch der Kinderheilkunde 351-355 (Opitz, H. et al., Berlin: Springer (pub.), 1967). More specifically, this disease resembles VBD in that it comprises a progressive sclerosing bone dysplasia characterized by generalized osteosclerosis and hyperostosis of the skeleton, affecting mainly the skull and mandible thereby causing facial paralysis and hearing loss. In contrast to VBD, however, sclerosteosis is further characterized by gigantism and hand abnormalities.
The rare genetic mutation responsible for the sclerosteosis syndrome has been localized to the region of human chromosome 17 that encodes a novel member of the TGF-beta binding-protein family (one representative example of which is designated xe2x80x9chSOSTxe2x80x9d). In 1999, Balemans et al. assigned the locus for sclerosteosis to 17q12-q21 which is the same general region as the locus for VBD. Am. J. Hum. Genet. 64:1661-1669 (1999). Due to the clinical similarities between VBD and sclerosteosis, Beighton et al. suggested that these conditions might be caused by mutations within the same gene. Clin. Genet 25:175-181 (1984). This hypothesis was further supported by the genetic experimentation later performed by Balemans et al. Supra.
Traditional methodologies for identifying genomic deletions such as, for example, restriction fragment length polymorphism (RFLP), fluorescence in situ hybridization (FISH) and Southern blotting permit the identification of individuals who are homozygous for a genomic deletion and, as a consequence, are afflicted with the associated genetic disease. Because these methods are time consuming and/or require high-quality DNA samples or live cells, they are of limited use in the identification of individuals who are heterozygous for and, therefore, carriers of a genetic disease. What is needed in the art are methods that permit the rapid identification of genetic disease carriers, which methods distinguish between individuals who are homozygous for a genomic deletion, individuals who are heterozygous for a genomic deletion and individuals who do not possess a given genomic deletion. As described in detail herein, the present invention fulfills this and other related needs.
The present invention is directed generally to disease diagnosis and to the identification of disease carriers. More specifically, the invention disclosed herein provides methods for identifying chromosomal deletions that are associated with a disease phenotype. Particular methods within the scope of the present invention are directed to the identification of individuals who are afflicted with or carriers for the genomic deletion associated with Van Buchem""s disease. By alternate embodiments, the present invention also provides methods having general utility in the detection of a wide variety of diseases characterized by genomic deletions.
The present invention provides, in one embodiment, methods for distinguishing between an individual who is homozygous for a genomic deletion, an individual who is heterozygous for a genomic deletion and an individual who is negative for a genomic deletion.
A first method comprises: (a) obtaining a sample of genomic DNA from an individual; (b) performing a first amplification reaction with a first oligonucleotide primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer wherein the first oligonucleotide primer is complementary to the nucleotide sequence upstream of the genomic deletion and the second oligonucleotide primer is complementary to the nucleotide sequence downstream of said genomic deletion; (c) performing a second amplification reaction with a second oligonucleotide primer pair comprising a third oligonucleotide primer and a fourth oligonucleotide primer wherein the third oligonucleotide primer is complementary to the nucleotide sequence either upstream or downstream of the genomic deletion and the fourth oligonucleotide primer is complementary to the nucleotide sequence comprising the genomic deletion; and (d) detecting the product of the amplification reactions of (b) and (c).
By this first type method, a positive amplification reaction of (b) and a negative amplification reaction of (c) indicates an individual who is homozygous for the large genomic deletion; a positive amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual who is heterozygous for the large genomic deletion; and a negative amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual who is negative for the large genomic deletion.
A second method comprises: (a) obtaining a sample of genomic DNA from said individual; (b) performing an amplification reaction employing at least two oligonucleotide primer pairs in which an oligonucleotide primer is common to both said primer pairs, wherein a first primer pair has a first oligonucleotide primer complementary to a nucleotide sequence that flanks said genomic deletion upstream of said genomic deletion and a second oligonucleotide primer complementary to a nucleotide sequence that flanks said genomic deletion downstream of said genomic deletion, and a second primer pair has third oligonucleotide primer complementary to a nucleotide sequence within said genomic deletion and either said first or second oligonucleotide primer; and (c) detecting an amplified product of said amplification reaction.
By this second method, a positive amplification reaction of said first primer pair and a negative amplification reaction of said second primer pair indicates an individual that is homozygous for said large genomic deletion; a positive amplification reaction of said first primer and a positive amplification reaction of said second primer pair indicates an individual that is heterozygous for said large genomic deletion; and a negative amplification reaction of said first primer pair and a positive amplification reaction of said second primer pair indicates an individual that is negative for said large genomic deletion.
Both these methodologies will find utility in the detection of large genomic deletions comprising at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50 kb. By some embodiments, the presence of the large genomic deletion is indicative of an individual who is either afflicted with or a carrier of a genetic disease. The genetic disease is exemplified herein by Van Buchem""s disease, but the methods are suitable for any disease characterized by large genomic deletions.
In further embodiments, the present invention provides a first method for identifying individuals who are afflicted with or carriers of Van Buchem""s disease. This first method comprises (a) obtaining a sample of genomic DNA from an individual; (b) performing a first amplification reaction with a first oligonucleotide pair primer having a first oligonucleotide primer and a second oligonucleotide primer wherein the first oligonucleotide primer is complementary to the nucleotide sequence upstream of the 51,719 bp sequence depicted in SEQ ID NO:2 which sequence corresponds to nucleotides 5,798 through 57,516 of the 92,149 bp nucleic acid sequence depicted in SEQ ID NO:1 and the second oligonucleotide primer is complementary to the nucleotide sequence downstream of the 51,719 bp sequence depicted in SEQ ID NO:2; and (c) performing a second amplification reaction with a second oligonucleotide pair primer comprising a third oligonucleotide primer and a fourth oligonucleotide primer wherein the third oligonucleotide primer is complementary to the nucleotide sequence either upstream or downstream of the 51,719 bp sequence depicted in SEQ ID NO:2 and the fourth oligonucleotide is complementary to the nucleotide sequence within the 51,719 bp sequence depicted in SEQ ID NO:2; and (d) detecting the product of the amplification reactions of (b) and (c).
By this first method, a positive amplification reaction of (b) and a negative amplification reaction of (c) indicates an individual afflicted with Van Buchem""s disease; a positive amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual who is a carrier of Van Buchem""s disease; and a negative amplification reaction of (b) and a positive amplification reaction of (c) indicates an individual that is neither afflicted with nor a carrier of Van Buchem""s disease.
Exemplary first oligonucleotide primer pairs may be selected from the group consisting of 12952/VBspan1 (SEQ ID NO:84/SEQ ID NO:85), Span1F/Span1R (SEQ ID NO:86/SEQ ID NO:87), Span2F/Span2R (SEQ ID NO:88/SEQ ID NO:89) and Vbspan2/Vbspan1 (SEQ ID NO:104/SEQ ID NO:85). Exemplary second oligonucleotide pairs may be selected from the group consisting of 12952/Wt1R (SEQ ID NO:84/SEQ ID NO:90), Wt2F/Wt2R (SEQ ID NO:91/SEQ ID NO:92), Wt3F/Wt3R (SEQ ID NO:93/SEQ ID NO:94) and VBspan2/VBint1 (SEQ ID NO:105/SEQ ID NO:102).
A second method comprises: (a) obtaining a sample of genomic DNA from said individual; (b) performing a polymerase chain reaction employing at least two oligonucleotide primer pairs in which an oligonucleotide primer is common to both said primer pairs, wherein a first primer pair has a first oligonucleotide primer that is complementary to a nucleotide sequence upstream of the 51,719 bp sequence provided in SEQ ID NO:2 and a second oligonucleotide primer that is complementary to a nucleotide sequence downstream of the 51,719 bp sequence provided in SEQ ID NO:2, and a second primer pair has a third oligonucleotide primer that is complementary to a nucleotide sequence within said genomic deletion and either said first or second oligonucleotide primer; and (c)detecting an amplified product of said amplification reaction.
By this second method, a positive polymerase chain reaction of said first primer pair and a negative polymerase chain reaction of said second primer pair indicates an individual afflicted with Van Buchem""s disease; a positive polymerase chain reaction of said first primer pair and a positive polymerase chain reaction of said second primer pair indicates an individual that is a carrier of Van Buchem""s disease; and a negative polymerase chain reaction of said first primer pair and a positive polymerase chain reaction of said second primer pair indicates an individual that is neither afflicted with nor a carrier of Van Buchem""s disease.
Exemplary first oligonucleotide primer pair is selected from the group consisting of 12952/VBspan1 (SEQ ID NO:84/SEQ ID NO:85), Span1F/Span1R (SEQ ID NO:86/SEQ ID NO:87), Span2F/Span2R (SEQ ID NO:88/SEQ ID NO:89), Wt2F/VBspan1 (SEQ ID NO:91/SEQ ID NO:85) and VBspan2/VBspan1 (SEQ ID NO:104/SEQ ID NO:85).
Still further embodiments of the present invention provide alternative methods for identifying individuals afflicted with Van Buchem""s disease. Exemplary methods comprise the steps of performing an amplification reaction with a pair of oligonucleotides selected from the region between nucleotide 1 and nucleotide 51,719 of SEQ ID NO:2 wherein the absence of an amplification product indicates an individual homozygous for Van Buchem""s disease. Exemplary oligonucleotide pairs are selected from the group consisting of Del1F/Del1R (SEQ ID NO:95/SEQ ID NO:96), Del2F/Del2R (SEQ ID NO:97/SEQ ID NO:98), and Del3F/Del3R (SEQ ID NO:99/SEQ ID NO:100).
Other embodiments provide methods for identifying an individual who is homozygous for Van Buchem""s disease comprising the step of detecting a deletion in human chromosome 17 at 17q21 between nucleotide 5,798 and nucleotide 57,516 as depicted in SEQ ID NO:1.
By still other embodiments are provided methods for detecting an individual who is afflicted with or a carrier of Van Buchem""s disease which methods comprise detecting the nucleotide sequence spanning the deletion breakpoint as depicted in FIG. 2 and SEQ ID NO:101. By these methods, the presence of a deletion breakpoint indicates an individual who is either afflicted with or a carrier of Van Buchem""s disease. Exemplary nucleotide sequences spanning the deletion breakpoint comprise the nucleotide sequence 5xe2x80x2-ACCATGCCCGGCTAAT-3xe2x80x2 (SEQ ID NO:102); the nucleotide sequence 5xe2x80x2-CTACCATGCCCGGCTAATTTT-3xe2x80x2 (SEQ ID NO:103); and the nucleotide sequence 5xe2x80x2-TGGGATTACAGGTGCATGCTACCATGCCCGGCTAATTTTTTTGTA TTTTTTTAGTA-3xe2x80x2 (SEQ ID NO:101).
In still further embodiments of the present invention are provided isolated polynucleotides. Preferred polynucleotides comprise at least 10, 15, 20, 25, 30, 40, 50, 100, 250 or 500 contiguous nucleotides of the nucleic acid depicted in SEQ ID NO:1. Alternatively, isolated polynucleotides according to the present invention hybridize under moderately stringent conditions to the nucleic acid depicted in SEQ ID NO:1 or the complement thereof. Preferred moderately stringent conditions comprise 2xc3x97SSC, 0.1% SDS at 65xc2x0 C.
Other embodiments provide isolated polynucleotides that comprise at least 10, 15, 20, 25, 30, 40, 50, 100, 250 or 500 nucleotides of one of the amplicons comprising the predicted exons within human chromosome 17 at 17q21 which amplicons are selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.
Still further embodiments of the present invention provide diagnostic kits for distinguishing between an individual who is homozygous for a large genomic deletion, an individual who is heterozygous for a large genomic deletion and an individual who is negative for a large genomic deletion.
A first kit comprises: (a) a first oligonucleotide primer pair comprising a first oligonucleotide primer and a second oligonucleotide primer, wherein said first oligonucleotide primer is complimentary to a nucleotide sequence upstream of said genomic deletion and said second oligonucleotide primer is complementary to a nucleotide sequence downstream of said genomic deletion; and (b) a second oligonucleotide primer pair comprising a third oligonucleotide primer and a fourth oligonucleotide primer wherein said third oligonucleotide primer is complementary to a nucleotide sequence either upstream or downstream of said genomic deletion and said fourth oligonucleotide primer is complementary to a nucleotide sequence within said genomic deletion. These types of diagnostic kit can further comprises instructions for distinguishing between an individual who is homozygous for a large genomic deletion, an individual who is heterozygous for a large genomic deletion and an individual who is negative for a large genomic deletion.
A second kit comprises: (a) a first primer pair having a first oligonucleotide primer that is complementary to a nucleotide sequence that flanks said genomic deletion upstream of said genomic deletion and a second oligonucleotide primer that is complementary to a nucleotide sequence that flanks said genomic deletion downstream of said genomic deletion; and (b) a second primer pair having a third oligonucleotide primer that is complementary to a nucleotide sequence within said genomic deletion and either said first or second oligonucleotide primer.
The genomic deletion mentioned can be associated with Van Buchem""s disease. In which case one type of diagnostic kit for identifying a carrier of Van Buchem""s disease comprises: (a) a first oligonucleotide primer pair comprising a first oligonucleotide and a second oligonucleotide wherein said first oligonucleotide is complementary to a nucleotide sequence upstream of the 51,719 bp sequence depicted in SEQ ID NO:2 and said second oligonucleotide is complementary to a nucleotide sequence downstream of the 51,719 bp sequence depicted in SEQ ID NO:2; and (b) a second oligonucleotide primer pair comprising a third oligonucleotide and a fourth oligonucleotide wherein said third oligonucleotide is complementary to either the nucleotide sequence upstream or downstream of said 51,719 bp sequence depicted in SEQ ID NO:2 and said fourth oligonucleotide is complementary to a nucleotide sequence within said 51,719 bp sequence depicted in SEQ ID NO:2. These diagnostic kits can further comprise instructions for identifying a carrier of Van Buchem""s disease.
A second kit comprises: (a) a first oligonucleotide pair having a first oligonucleotide primer that is complementary to a nucleotide sequence upstream of the 51,719 bp sequence provided in SEQ ID NO:2 and a second oligonucleotide primer that is complementary to a nucleotide sequence downstream of the 51,719 bp sequence depicted in SEQ ID NO:2; and (b) a second oligonucleotide pair having a third oligonucleotide primer that is complementary to a nucleotide sequence within said 51,719 bp sequence provided in SEQ ID NO:2 and either said first or second oligonucleotide primer.